Maternal separation facilitates extinction of social fear in adult male mice

Behavioural Brain Research 297 (2016) 323–328

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Maternal separation facilitates extinction of social fear in adult male mice Iulia Zoicas 1 , Inga D. Neumann ∗ a

Faculty of Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany

h i g h l i g h t s • • • •

We use maternal separation (MS, on postnatal days 1–14) to induce early life stress. We use social fear conditioning to induce social fear in adult MS and control mice. Maternal separation facilitates extinction of social, but not of non-social fear. Early life stress improves recovery from a traumatic social experience in adulthood.

a r t i c l e

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Article history: Received 14 July 2015 Received in revised form 28 September 2015 Accepted 15 October 2015 Available online 20 October 2015 Keywords: Early life stress Social fear conditioning Cued fear conditioning Social memory Non-social memory Anxiety

a b s t r a c t Early life stress, such as child abuse or neglect, is a risk factor for the development of psychopathologies characterized by abnormal social and emotional behaviors. In rodents, long-lasting changes in stress coping and emotional behavior can be induced by separating pups from their mother. We used maternal separation (MS; 3 h daily on postnatal days 1–14) to test whether early life stress alters acquisition and extinction of social fear in adult male mice as studied in a specific model of social fear, i.e., in the social fear conditioning paradigm. We show that MS facilitated extinction of social fear without altering acquisition or expression of social fear. This facilitatory effect of MS on social fear extinction was not due to improved social learning and memory abilities or to increased social interest, as MS rather impaired social memory in the social discrimination test and did not alter social preference in the social preference-avoidance test. In contrast, MS did not alter acquisition and extinction of non-social, cued fear, or non-social memory as assessed in the object discrimination test and non-social anxiety as assessed in the elevated plus-maze. These results suggest that a social stress like MS in early life may improve coping with and recovery from a traumatic social experience in adulthood in mice. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Epidemiologic studies indicate that children exposed to early life adversities, such as child abuse or neglect, have an increased risk of developing psychopathologies characterized by inappropriate social and emotional behaviors, such as anxiety disorders, major depression, alcohol and drug abuse [1,2]. Maternal separation (MS) is a paradigm, which has been used to mimic child maltreatment conditions in rodents [3,4] and was shown to induce long-lasting

∗ Corresponding author at: University of Regensburg, Department of Behavioral and Molecular Neurobiology, Universitaetsstrasse 31, 93053 Regensburg, Germany. Fax: +49 9419433052. E-mail address: [email protected] (I.D. Neumann). 1 Present address: Department of Psychiatry and Psychotherapy, University Hospital, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany. http://dx.doi.org/10.1016/j.bbr.2015.10.034 0166-4328/© 2015 Elsevier B.V. All rights reserved.

changes in stress coping, as well as emotional and social behaviors in rats [5,6] and mice [7–9]. As such, separating pups from their mother for 3 h daily on postnatal days 1–14 increases offensive-like behaviors during juvenile play-fighting [9] and aggression during adult resident–intruder encounters [8,10], suggesting that MS alters coping with social conflict situations throughout life. However, it is unclear, whether MS also alters coping with and recovery from a traumatic social experience in adulthood. In order to induce long-lasting social fear in adult MS and control mice, we used the social fear conditioning (SFC) paradigm, which involves administration of mild electric foot-shocks during the investigation of a con-specific and results in reduced social investigation and avoidance of con-specifics, as indicative of social fear [11,12]. Repeated exposure of the socially fear-conditioned (SFC+ ) mice to unknown con-specifics leads to a gradual decline in the fear response, a process termed social fear extinction.

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In the present study, we used the SFC paradigm to investigate the effects of early life stress on acquisition, expression and extinction of social fear in adult male mice. As MS facilitated extinction of social fear, but did not alter acquisition and expression of social fear, we also assessed the effects of MS on acquisition, expression and extinction of non-social fear by using the cued fear conditioning (CFC) paradigm [13,14]. Further, we assessed the effects of MS on social and non-social memory and anxiety. 2. Materials and methods 2.1. Animals Male and female CD1 mice were obtained from Charles River (Sulzfeld, Germany) and kept under standard laboratory conditions (12:12 light/dark cycle, lights on at 06:00 h, 22 ◦ C, 60% humidity, food and water ad libitum). Pregnant females were group-housed after 5 days of mating. During the last week of gestation, females were individually housed in standard mouse cages (16 × 22 × 14 cm). Experiments were performed during the light phase, between 08:00 and 12:00, in accordance with the Guide for the Care and Use of Laboratory Animals of the Government of Oberpfalz and the guidelines of the NIH. 2.2. Maternal separation (MS) procedure Early life stress was induced by MS as previously described [10]. Briefly, on the day after parturition, i.e., on postnatal day 1, each litter was culled to 8 pups (2–4 females in each nest). Litters were separated from the mother daily between 09:00 and 12:00 from postnatal day 1–14. Each dam was placed into a clean cage and the litter was transferred to an adjacent room and kept in a box filled with bedding and placed on a heating pad maintained at 30–33 ◦ C. After 3 h, litters were returned to the home cage followed by the dam. Control litters were left undisturbed. Change of bedding occurred on postnatal day 1, 7 and 14 for both control and MS litters. Male pups were weaned on postnatal day 21 and housed in groups of 6. Experiments were performed between 9 and 12 weeks of age. 2.3. Social fear conditioning (SFC) paradigm The acquisition and extinction of social fear was assessed in the SFC paradigm as previously described [11,12] using a computerized fear conditioning system (TSE System GmbH, Bad Homburg, Germany). 2.3.1. SFC (day 1) Mice were placed in the conditioning chamber (45 × 22 × 40 cm) and, after a 30 s habituation period, an empty wire mesh cage (7 × 7 × 6 cm) was placed as a non-social stimulus near one of the short walls. After 3 min, the non-social stimulus was replaced by an identical cage containing an unfamiliar age- and weight-matched male mouse. Unconditioned mice (SFC− ) were allowed to investigate this social stimulus for 3 min, whereas conditioned mice (SFC+ ) were given a 1 s electric foot shock (0.7 mA) each time they investigated, i.e., made direct contact with the social stimulus. Mice received between 1 and 3 foot shocks, with a variable inter-shock interval, depending on when direct social contact was made. Mice were returned to their home cage, when no further social contact was made for 2 min. The time mice spent investigating the non-social stimulus, as a pre-conditioning measure of non-social anxiety, was analyzed with the JWatcher program (V 1.0, Macquarie University and UCLA).

2.3.2. Social fear extinction (day 2) One day after SFC, mice were exposed in their home cage to 3 non-social stimuli, i.e., empty cages identical to the cage used on day 1, to assess non-social investigation as a parameter of nonsocial fear. Mice were then exposed to 6 different unfamiliar social stimuli, i.e., male mice enclosed in wire mesh cages, to assess social investigation as a parameter of social fear. Each stimulus was placed near a short wall of the home cage and presented for 3 min, with a 3 min inter-exposure interval. 2.3.3. Extinction recall (day 3) One day after social fear extinction, mice were exposed in their home cage to 6 unfamiliar social stimuli for 3 min, with a 3 min inter-exposure interval. 2.4. Cued fear conditioning (CFC) paradigm The acquisition and extinction of non-social fear was assessed in the CFC paradigm as previously described [14,15]. Two different contexts, which differed in visual, tactile, and olfactory cues, were used. The time displaying freezing, as an indicator of cued fear, was assessed with the TSE System. 2.4.1. CFC (day 1) Mice were placed in context A (transparent Perspex box with an electric grid floor, cleaned with neutral-smelling detergent) and, after a 5 min habituation period, exposed to 5 conditioned stimulus—unconditioned stimulus (CS–US) pairings, with 2 min inter-stimulus intervals. The CS was an 80-dB, 8-kHz, 30 s white noise, which co-terminated with an electric foot shock (US; 0.7 mA, pulsed current, 2 s). Mice were returned to their home cage 5 min after the last CS–US pairing. 2.4.2. Cued fear extinction (day 2) One day after CFC, mice were placed in context B (black Perspex box with a smooth floor, cleaned with lemon-scented detergent) and, after a 5 min habituation period, exposed to 20 CS presentations, with 5 s inter-stimulus intervals. Mice were returned to their home cage 5 min after the last CS presentation. The mean freezing percentage during two consecutive CS presentations was collapsed into one block. 2.4.3. Extinction recall (day 3) One day after cued fear extinction, mice were placed in context B and, after a 5 min habituation period, exposed to 5 CS presentations, with 5 s inter-stimulus intervals. Mice were returned to their home cage after the last CS presentation. The mean freezing percentage during these 5 CS presentations was collapsed into one block. 2.5. Social discrimination test Social memory was assessed in the social discrimination test as previously described [16]. Mice were exposed in their home cage to a 3-week-old male CD1 mouse for 4 min (acquisition period). After an 1 h inter-exposure interval (IEI), during which mice and rats typically maintain social memory [16], mice were exposed to the same juvenile along with a novel juvenile for 4 min (social discrimination period). An increased investigation of the novel versus the same juvenile indicated social discrimination and intact social memory. 2.6. Object discrimination test Object memory was assessed in the object discrimination test as previously described [16]. Mice were exposed in their home cage to an object for 4 min (acquisition period). After a 2 h or 4 h IEI, mice

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Fig. 1. Maternal separation facilitates extinction of social fear in male mice. Pre-conditioning investigation of the non-social stimulus by maternally-separated (MS) and control unconditioned (SFC− ) and conditioned (SFC+ ) mice during social fear conditioning (A). Investigation of the non-social (ns1–ns3; 3 different empty cages) and social (s1–s6; 6 different con-specifics) stimuli during social fear extinction (B) and extinction recall (C). Data represent means ± SEM. * p < 0.05 vs MS SFC+ mice; #p < 0.05 vs control SFC− and MS SFC− mice. Table 1 Overall statistics for the behavioral data. MS, maternal separation; SFC, social fear conditioning; CSF, cued fear conditioning; SPAT, social preference-avoidance test; EPM, elevated plus-maze. Student’s t-test, one-way or two-way ANOVA for repeated measures followed by Bonferroni post-hoc test; * p < 0.05. Effects of MS on social fear (n = 8–9 mice/group)

SFC (Fig. 1A) Social fear extinction (Fig. 1B) Extinction recall (Fig. 1C)

Group effect F(3,31) = 0.20; p = 0.90 F(3,31) = 12.09; p < 0.001* F(3,31) = 2.73;p = 0.06

Group x stimulus effect

Group effect F(1,22) = 0.37; p = 0.55 F(1,22) = 0.22; p = 0.65 T(22) = 0.70; p = 0.50

Group x CS effect F(4,88) = 0.08; p = 0.99 F(9198) = 1.47; p = 0.16

F(24,248) = 10.51; p < 0.001* F(15,155) = 1.01; p = 0.45

Effects of MS on non-social, cued fear (n = 12 mice/group)

CFC (Fig. 2A) Cued fear extinction (Fig. 2B) Extinction recall (Fig. 2C)

Effects of MS on social and non-social, object memory (n = 12 mice/group)

Social memory, 1 h IEI (Fig. 3A) Object memory, 2 h IEI (Fig. 3B) Object memory, 4 h IEI (Fig. 3C)

Group effect F(1,48) = 0.0; p = 1.0 F(1,48) = 0.0; p = 1.0 F(1,48) = 0.0; p = 1.0

Stimulus effect F(1,48) = 13.29; p = 0.001* F(1,48) = 12.02; p = 0.001* F(1,48) = 10.54; p = 0.002*

Effects of MS on social and non-social anxiety (n = 12 mice/group)

SPAT (Fig. 4A) EPM: time open arms (Fig. 4B) EPM: closed arm entries (Fig. 4C)

Group effect F(1,48) = 5.21; p = 0.03* T(22) = 0.54; p = 0.60 T(22) = 1.01; p = 0.32

were exposed to the same object along with a novel object for 4 min (object discrimination period). Several plastic objects that differed in color (pink, green, orange), shape (flower, diamond, square), and size (3–4.5 × 3 × 1 cm) were used to prevent potential object preference between mice. An increased investigation of the novel versus the same object indicated object discrimination and intact nonsocial memory. A 2 h and 4 h IEI was used because object memory is maintained for at least 2 h in rats [37].

Stimulus effect F(1,48) = 45.18; p < 0.001*

2.8. Elevated plus-maze (EPM) Non-social anxiety was assessed on the EPM as previously described [11,14]. Increased percentage of time spent on the open arms (110 lx) indicated reduced anxiety. The number of entries into the closed arms (25 lx) during the 5 min testing period indicated locomotor activity. 2.9. Statistical analysis

2.7. Social preference-avoidance test (SPAT) Social anxiety was assessed in the SPAT as previously described [17]. Mice were placed in a novel arena (28 × 46 × 27 cm; 100 lx) and, after a 30 s habituation period, an empty wire mesh cage (7 × 7 × 6 cm) was placed as a non-social stimulus near one of the short walls for 2.5 min. This non-social stimulus was then replaced by an identical cage containing an unfamiliar age- and weightmatched male mouse for additional 2.5 min. An increased social versus non-social investigation indicated social preference and, thus, a lack of social anxiety, whereas a decreased social versus non-social investigation indicated social avoidance and, thus, social anxiety.

For statistical analysis PASW/SPSS (Version 17) was used. Data were analyzed by Student’s t-test, one-way or two-way ANOVA for repeated measures, followed by a Bonferroni’s post-hoc analysis whenever appropriate. Statistical significance was set at p < 0.05. Overall statistics are shown in Table 1. 3. Results 3.1. MS facilitates extinction of social fear To investigate whether MS affects acquisition and extinction of social fear, MS and control mice (n = 8–9/group) were tested in the SFC paradigm.

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Fig. 2. Maternal separation does not affect acquisition, expression and extinction of non-social, cued fear in male mice. Conditioned stimulus (CS)-induced freezing during cued fear conditioning (A), cued fear extinction (B) and extinction recall (C) in maternally-separated (MS) and control mice. Data represent means ± SEM.

Fig. 3. Maternal separation (MS) impairs social, but not non-social memory in male mice. Investigation of the same and the novel juvenile (A) or object (B,C) during the social and object discrimination period, respectively. Intact memory and discrimination ability are reflected by an increased investigation of the novel versus the same stimulus after an inter-exposure interval of 1 h (A), 2 h (B) or 4 h (C). Data represent means ± SEM. * p < 0.05 vs same stimulus.

On day 1, all mice showed similar non-social investigation during SFC (Fig. 1A; Table 1), indicating similar pre-conditioning non-social anxiety. MS SFC+ and control SFC+ mice received a similar number of foot shocks during SFC (MS 2.13 ± 0.3; control 2.39 ± 0.16; T(15) = 0.81; p = 0.43), indicating similar distress. During social fear extinction on day 2, all mice showed similar non-social investigation (Fig. 1B). Although both MS SFC+ and control SFC+ mice showed reduced investigation of the first social stimulus, indicating comparable acquisition and expression of social fear (Fig. 1B), extinction of social fear was facilitated in MS mice. In more detail, while in control SFC+ mice social investigation was reduced during exposure to the first 5 social stimuli (p < 0.05 vs SFC− groups), in MS SFC+ mice social investigation was reduced only during exposure to the first 2 social stimuli and completely returned to levels found in SFC− mice starting from the 3rd social stimulus. During extinction recall, all mice showed similar social investigation (Fig. 1C).

3.2. MS does not affect acquisition and extinction of non-social, cued fear To investigate whether MS also facilitates extinction of nonsocial, cued fear, separate groups of MS and control mice (n = 12/group) were tested in the CFC paradigm. There was no significant difference between MS and control mice during CFC, cued fear extinction and extinction recall (Fig. 2).

3.3. MS impairs social, but not non-social, object memory To investigate whether the facilitated extinction of social fear in MS mice might be due to altered social and/or non-social memory abilities, separate groups of MS and control mice (n = 12/group) were tested in the social and object discrimination tests. While control mice demonstrated social memory abilities after an IEI of 1 h, as indicated by an increased investigation of the novel versus the same juvenile (p < 0.05) during the social discrimination period, social memory was impaired in MS mice (Fig. 3A). In contrast, non-social memory was not altered by MS, as indicated by an increased investigation of the novel versus the same object during the object discrimination period in both control and MS mice even after an IEI of 2 h and 4 h (p < 0.05; Fig. 3B,C).

3.4. MS does not alter social and non-social anxiety To investigate whether the facilitated extinction of social fear in MS mice might be due to altered social and/or non-social anxiety, separate groups of MS and control mice (n = 12/group) were tested in the SPAT and on the EPM. Both control and MS mice showed naturally occurring social preference and, thus, a lack of social anxiety in the SPAT, as indicated by an increased investigation of the social versus the non-social stimulus (p < 0.05; Fig. 4A). Similarly, MS did not alter non-social anxiety and locomotor activity on the EPM, as

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Fig. 4. Maternal separation does not alter social and non-social anxiety in male mice. Investigation of the non-social (empty cage) and social (cage + mouse) stimulus during the social preference-avoidance test (A) in maternally-separated (MS) and control mice. Time spent on the open arms (B) and number of entries into the closed arms (C) of the elevated plus-maze. Data represent means ± SEM. * p < 0.05 vs empty cage.

indicated by similar time spent on the open arms and similar number of closed arm entries, respectively, between control and MS mice (Fig. 4B,C). 4. Discussion To the best of our knowledge, this is the first study to report a facilitatory effect of repeated MS on extinction of social fear and suggests that social stress early in life improves coping with and recovery from a traumatic social experience in adulthood. Interestingly, MS did not alter acquisition and expression of social fear, suggesting that the facilitated social fear extinction is unlikely due to an impaired ability to learn or to express social fear. Similarly, MS did not alter the ability to learn or to express non-social, cued fear, which partly supports previous findings. As such, acquisition of cued fear was either slightly impaired [18] or unaltered [19] in MS rats, while expression of cued fear was either impaired in BALB/cJ mice [20] or unaltered in rats [18] after MS. These discrepancies may directly relate to the conditioning procedure, i.e., the different number, intensity and duration of foot-shocks used in these studies. In contrast to social fear, however, MS did not affect the extinction of cued fear, supporting previous studies in male rats [18,19]. Together, these data show that MS specifically facilitates extinction of social, but not of non-social, fear and suggest that a social stress like MS in early life improves coping with and recovery from a traumatic social experience in adulthood. As MS did not affect the number of foot-shocks received during SFC and the amount of freezing during CFC, it is unlikely that the facilitatory effect on social fear extinction is due to altered pain sensitivity in MS mice. In support, 3 h of daily MS from postnatal day 1–14 in male rats did not alter pain sensitivity to foot-shock intensities ranging from 0.4 mA to 3.2 mA, as indicated by similar vocalization, limb withdrawal and jumping during the foot-shocks [21]. However, MS was previously shown to decrease sensitivity to noxious thermal stimuli [22] and to increase visceral pain sensitivity [23,24]. We have previously shown that MS impairs social memory in male rats, i.e., the recognition of a previously encountered conspecific after an IEI of 60 min [16], and could now confirm this effect in male mice. In contrast, object recognition was unaltered by MS even after an IEI of 2 h and 4 h as demonstrated in the object discrimination test, indicating that MS specifically impairs social, but not non-social, memory. However, the impaired ability of MS mice to recognize individual con-specifics does not seem to

interfere with their ability to remember a social trauma like SFC, as indicated by similar expression of social fear between MS and control mice. This might be due to the fact that SFC+ mice fear both previously encountered and unknown con-specifics [11]. By exposing SFC+ mice only to unknown con-specifics during the social fear extinction procedure, recognition of a previously encountered con-specific becomes unnecessary in the SFC paradigm. Another possibility is the recruitment of different brain regions to encode the information necessary for social memory and social fear. While brain regions like the olfactory bulb [25], lateral septum [16,26], medial amygdala, bed nucleus of the stria terminalis, and medial preoptic area [26] are recruited for the recognition of a previously encountered con-specific, additional brain regions like the central amygdala and the hippocampus [27] are recruited for the expression of social fear. Our finding that social preference as assessed in the SPAT is not altered in MS mice confirms previous studies in mice [28] and rats [29] and indicates that the facilitatory effect of MS on social fear extinction in unlikely due to decreased social anxiety. The effects of MS on non-social anxiety, however, are more controversial in both mice and rats. Studies in MS rats demonstrate either increased [5,30], decreased [31], or no changes in non-social anxiety [29] as assessed in the EPM, open field test and light–dark box. Similarly, MS increased non-social anxiety in male C57BL/6 mice, but not in female C57BL/6 or male CD1 mice [7,32,33], supporting our findings in male CD1 mice. A study performed in both males and females of eight inbred strains of mice (129S1/SvImJ, 129P3/J, A/J, BALB/cJ, BALB/cByJ C57BL/6J, DBA/2J, FVB/NJ) showed that MS does not alter non-social anxiety in these strains as assessed in the EPM and light–dark box [34]. This variability in non-social anxiety may in part relate to methodological differences, but also to the modifying influence of genetic factors such as innate anxiety and sensitivity to various stressors [34]. Similar to our results showing improved coping with a social stress after MS, it was shown that MS can also have positive behavioral and neuroendocrine effects depending on the level of innate anxiety [35]. As such, in rats selectively bred for extremes in trait anxiety, i.e., high (HAB) or low (LAB) anxiety-related behavior, MS decreased anxiety in HAB rats and increased anxiety in LAB rats as assessed in the modified holeboard and EPM. Furthermore, the hyper-responsiveness of the hypothalamic–pituitary–adrenal axis which accompanies the high anxiety in HAB rats [36] was attenuated by MS, as indicated by reduced ACTH and corticosterone responses to an acute stress in MS compared with control HAB

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rats [35]. These studies suggest that new-born animals are prepared to cope optimally with the environment in which they are born, long before they start learning from their own (and often dangerous) experiences. Taken together, we have shown that MS specifically facilitates extinction of social fear and that this facilitatory effect is unlikely due to changes in social memory or social anxiety. On the other hand, MS did not alter non-social, cued fear extinction, non-social, object memory or non-social anxiety. Our study suggests that a social stress like MS in early life may improve coping with and recovery from a traumatic social experience in adulthood.

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IZ received financial support from the Bayerische Forschungsstiftung. IDN received financial support from BMBF and Deutsche Forschungsgemeinschaft (Ne465/23).

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